1. Field of the Invention
This invention relates to a DNA base sequence which contains regions involved in the production and secretion of a protein, a recombinant DNA which includes the whole or a part of the DNA base sequence, and a method of producing proteins which comprises introducing the recombinant DNA into a microorganism, culturing this microorganism and thereby causing the protein to be extracellularly secreted in large amounts, and then recovering the protein.
2. Description of the Prior Act
Bacteria of the genus Bacillus are known to secrete a variety of proteins extracellularly. Among others, neutral protease is one of the most useful enzymes that are currently produced on an industrial scale, and its wide field of application covers, for example, the manufacture of foods and cosmetics, the tanning of hides and the production of dry cleaning soap. Conventionally, neutral protease has been obtained by culturing a bacterium which is highly productive of this enzyme (such as B. amyloliquefaciens, B. subtilis, B. sacchariticus or B. licheniformis), collecting the cell-free culture medium which contains neutral protease, and separating the enzyme into a purified form. However, in addition to neutral protease, Bacillus bacteria secrete and accumulate a large amount of other extracellular proteins (such as amylase, alkaline protease and levansucrase) in the cell-free culture medium. Accordingly, in the production of neutral protease, much effort has been required to remove such contaminants.
As an effective means of overcoming this disadvantage, attention is being given to the microbial production of proteins by utilizing the cloning technique in which a gene coding for a desired protein is combined with a suitable vector and the resulting recombinant DNA is introduced into a host bacterium to transform it.
More specifically, a DNA fragment which contains regions involved in the production and secretion of a desired protein is isolated and combined with a suitable vector to form a recombinant DNA which permits the genetic information concerning the production and secretion of the protein to be expressed in host cells. Then, this recombinant DNA is introduced into a suitable host bacterium, which is cultured to cause the protein to be secreted in large amounts. Thus, the protein can be recovered from the cell-free culture medium according to a simple procedure.
The DNA fragment used for forming the recombinant DNA obtained by the above-described method must contain regions which are involved in the production and secretion of the desired protein, that is, the promoter region, the ribosome binding region, the structural gene for the desired protein, and the terminator region.
The regions involved in the expression of the gene include the promoter region which has the -35 and -10 regions which act as RNA polymerase recognition and binding sites; and the ribosome binding region which defines the base sequence through which the messenger RNA synthesized by RNA polymerase binds ribosomes. The base sequences of these regions are very important for the efficiency of the gene expression. In addition, the distance (i.e., the number of bases) between these regions 15 also known to be very important [Moran, Jr., et al., Mol. Gen. Genet., 186, 339(1982)].
In the extracellular secretion of the protein synthesized in the cells as a result of gene expression, the region which codes for the polypeptide chain upstream of the amino end of the protein secreted extracellularly as mature protein is important. The protein newly synthesized in the cells has this polypeptide chain in the form combined with the amino end of the mature protein, but as soon as the polypeptide chain is removed by the action of peptidase, the newly synthesized protein is extracellularly secreted to provide mature protein. Accordingly, the region of the gene which codes for the polypeptide chain is essential to the secretion of the protein synthesized as a result of gene expression.
Moreover, it is desirable from the viewpoint of the industrial production of proteins that the DNA fragment which is used for forming the recombinant DNA have the characteristics which permit a high-level expression of the gene and efficient secretion of the protein.
Thus, in the industrial production of proteins which use the genetic engineering techniques explained thus far, it is very important to clone a DNA fragment which is a chromosome segment which has the structural gene for a desired protein and regions which are involved in the expression of the gene and the secretion of the resulting protein, to isolate the DNA fragment in a pure form, determine its DNA base sequence, and apply it to practical uses.
Bacteria of the genus Bacillus are preferred as host microorganisms from an industrial point of view, because they lack pathogenicity, can be easily handled and cultured, and have long been used in fermentation technology (Debabov, "The Molecular Biology of the Bacilli", 1 332(1982), Dubnau, D. A., ed., Academic Press).
However, where a foreign gene is to be expressed in host bacilli, the RNA polymerase and ribosomes of bacilli have rigid specificity in relation to the recognition of the promoter region and the ribosome binding region [Sueji Horinouchi, Tanpakushitsu-Kakusan-Koso, 28, 1468(1983)], so that these regions must be derived from bacilli [Goldfarb, D. S., et al., Nature, 293, 309(1981)].
From this point of view, an attempt has been made to produce foreign proteins by combining a foreign gene with a DNA base sequence which contains the promoter and ribosome binding regions specific for bacilli and allowing this recombinant DNA to be expressed in a host bacillus [Williams, D. M., et al., Gene, 16, 199 (1981)]. Moreover, an effort has also been made to obtain powerful promoter regions and signal sequences for extracellularly secreted enzymes of bacilli [Palva, I., et al., Gene, 15, 43 (1981)].
However, in preparing DNA fragments for use in the formation of recombinant DNA molecules which contain regions involved in the production and secretion of, for example, neutral protease and particularly its powerful promoter region and signal sequence, it becomes difficult to form recombinant DNA molecules which comprise DNA fragments combined with a vector if the size of the chromosome segments obtained by cleaving the chromosomes isolated from a donor microorganism exceeds a certain limit [Takagi Y., ed., "Experimental Methods for Gene Manipulation", p. 139, Kodansha (1980)]. Even if chromosome segments of such an exceedingly large size can be combined with a vector to form recombinant DNA molecules, these recombinant DNA molecules cannot be stably retained within host cells, so that it is impossible to obtain the desired DNA segment by cloning. Thus, it is necessary to select a restriction enzyme which can yield chromosome segments of proper size.
However, no restriction enzyme that is suitable for this purpose and which can be successfully used in combination with the chromosomes isolated from a donor microorganism is known as yet.
Even in the case of restriction enzymes which have a cleavage site in the structural gene for neutral protease or its promoter region or signal sequence, they can theoretically be used if the chromosomes are treated under partial cleavage conditions. However, if a plurality of such cleavage sites is present in the DNA fragment, the fragment will unavoidably be cleaved at some of those sites, thus making it difficult to clone the DNA fragment.